The present application is directed to inducing resistance to tumor growth by using diffusion chambers implanted in mammals.
Traditional methods of treating tumors in mammals include procedures such as, for example, surgical removal of the tumor, injection or implantation of toxic treatments or syngeneic tissue samples, chemotherapy, and irradiation. The ultimate goal of each of these procedures is to reduce the growth of existing tumors, preferably abrogating tumor growth to the point of complete regression, and/or to induce resistance to future tumor growth. These procedures have numerous effects on tumor cells.
Tumors and other neoplastic tissues are known to undergo apoptosis spontaneously or in response to treatment. Examples include several types of leukemia, non-Hodgkin""s lymphoma, prostate tumor, pancreatic cancer, basal and squamous cell carcinoma, mammary tumor, breast cancer, and fat pad sarcoma. Several anticancer drugs have been shown to induce apoptosis in target cells. Buttyan, et al., Mol. Cell. Biol., 1989, 9, 3473-3481; Kaufmann, Cancer Res., 1989, 49, 5870-5878; and Barry, et al., Biochem. Pharmacol., 1990, 40, 2353-2362, all of which are incorporated herein by reference. Certain mildly adverse conditions can result in the injured cell dying by programmed cell death, including hyperthermia, hypothermia, ischemia, and exposure to irradiation, toxins, and chemicals. It should be noted that many of these treatments will also result in necrosis at higher doses, suggesting that mild injury to a cell might induce cell suicide, perhaps to prevent the inheritance of a mutation, while exposure to severe conditions leads directly to cell death by necrosis. However, the death process is difficult to observe due to the rapidity of the process and the reduced amount of inflammation. For these reasons, quantification of apoptosis is often difficult. A method of measuring the duration of the histologically visible stages of apoptosis (3 hours in normal rat liver) and a formula by which to calculate the cell loss rate by apoptosis is set forth by Bursch, et al., Carcinogenesis, 1990, 11, 847-853.
Evidence is also rapidly accumulating that growth factors and their receptors play a crucial role in the establishment and maintenance of transformed phenotypes. It is well established that growth factors play a crucial role in the establishment and maintenance of the transformed phenotype. Mouse embryo cells with a targeted disruption of the type 1 insulin-like growth factor receptor (IGF-IR) genes cannot be transformed by SV40 T antigen and/or an activated Ha-ras oncogene that easily transform embryo cells generated from their wild-type littermates. Sell, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 11217-11221; Sell, et al., Mol. Cell. Biol., 1994, 14, 3604-3612; Valentinis, et al., Oncogene, 1994, 9, 825-831; and Coppola, et al., Mol. Cell. Biol., 1994, 14, 4588-4595. Expression of an antisense RNA to the IGF-IR RNA in C6 rat glioblastoma cells not only abrogates tumorigenesis in syngeneic rats, but also causes complete regression of established wild type tumors. Resnicoff, et al., Cancer Res., 1994a, 54, 2218-2222 and Resnicoff, et al., Cancer Res., 1994b, 54, 4848-4850. Related to this finding is also the report by Harrington, et al. (EMBO J., 1994, 13, 3286-3295), that IGF-I (and PDGF) protect cells from c-myc induced apoptosis. A decrease in cell death rate in tumors could certainly be an important mechanism for tumor growth. Baserga, The Biology of Cell Reproduction, Harvard University Press, Cambridge, Mass., 1985. Cells expressing an antisense RNA to the IGF-IR RNA or cells pre-incubated with antisense oligodeoxynucleotides to the IGF-IR RNA completely lose their tumorigenicity when injected in either syngeneic or nude mice. Resnicoff et al., 1994a, 1994b. The injected cells were suspected of undergoing apoptosis or, at any rate, some form of cell death. Dying cells, however, are very difficult to demonstrate, because dying cells, especially in vivo, disappear very rapidly, and one is left with nothing to examine.
The importance of the IGF-I receptor in the control of cell proliferation is also supported by considerable evidence: 1) many cell types in culture are stimulated to grow by IGF-I (Goldring, et al., Crit. Rev. Eukaryot. Gene Expr., 1991, 1, 301-326 and Baserga, et al., Crit. Rev. Eukaryot. Gene Expr., 1993, 3, 47-61), and these cell types include human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, myeloid cells, chondrocytes, osteoblasts as well as the stem cells of the bone marrow; 2) interference with the function of the IGF-I receptor leads to inhibition of cell growth. This has been demonstrated by using antisense expression vectors or antisense oligodeoxynucleotides to the IGF-I receptor RNA: the antisense strategy was successful in inhibiting cellular proliferation in several normal cell types and in human tumor cell lines (Baserga, et al., 1994, supra.); and 3) growth can also be inhibited using peptide analogues of IGF-I (Pietrzkowski, et al., Cell Growth and Diff., 1992a, 3, 199-205 and Pietrzkowski, et al., Mol. Cell. Biol., 1992b, 12, 3883-3889), or a vector expressing an antisense RNA to the IGF-I RNA (Trojan, et al., 1993, supra.). The IGF autocrine or paracrine loop is also involved in the growth promoting effect of other growth factors, hormones (for instance, growth hormone and estrogens), and oncogenes like SV40 T antigen and c-myb, and in tumor suppression, as in the case of WT1. Baserga, et al., 1994, supra.
Testing agents such as, for example, growth factors and growth factor receptors for their ability to maintain or suppress transformed phenotypes remains difficult. In order to obtain an accurate account of the tumor suppressive ability, testing should be performed in vivo. Therapies such as direct injection or implantation of toxic treatments, tissue samples, and chemotherapy often jeopardizes the overall health of the patient. However, the present invention provides a method of inducing resistance to tumor growth with markedly reduced side effects to the patient.
The present invention is directed to a method of inducing resistance to tumor growth in a mammal comprising pretreating tumor cells in vitro with a pro-apoptotic agent, placing the pretreated tumor cells in a diffusion chamber thereby producing a tumor cell-containing diffusion chamber, and inserting the tumor cell-containing diffusion chamber into the mammal for a therapeutically effective time thereby inducing resistance to tumor growth.
The present invention is also related to a method of screening test compounds for anti-cancer activity in a mammal comprising providing an in vitro tumor cell culture supplemented with a test compound, placing the tumor cells into a diffusion chamber thereby producing a tumor cell-containing diffusion chamber, inserting the tumor cell-containing diffusion chamber into the mammal for a period of time, and removing the tumor cell-containing diffusion chamber and evaluating the anti-cancer effects of the test compound.